The long-term objectives of this application are to understand the basic mechanisms of nuclear RNA processing in the yeast Saccharomyces cerevisiae. RNA processing is largely conserved between yeast and humans and many steps occur predominantly co-transcriptionally, including splicing and RNP assembly. Whereas some of these events are well-understood, others are not. Further investigation is needed to understand the mechanisms by which RNP proteins and splicing factors are recruited co-transcriptionally as well as the relationship between chromatin and completed transcripts, i.e., after 3 end formation but before nuclear export. A first goal is to continue our work on the early steps of splicing as well as the relationship of splicing and spliceosome assembly to transcription. Proteins that impact more general aspects of RNP function as well as splicing will be also be studied. A second goal is to characterize chromatin by mass spectrometry, with an emphasis on proteins involved in post-transcriptional events. The strategy will take advantage of yeast genetics and look at interesting and well-studied mutants, which should give rise to missing chromatin proteins. New relationships should be uncovered, i.e., new proteins involved in these defined biochemical processes. A third goal is to visualize active genes by light and electron microscopy. Genes will be separated from the rest of the chromosomal DNA by in vivo endonuclease cleavage and their movement within nuclei examined by light microscopy. Visualization of individual pot II genes will also be done by Miller spreading, with an emphasis on co-transcriptional spliceosome assembly and splicing. A fourth goal is to identify the function(s) of individual yeast RNA binding proteins, with an emphasis on nuclear proteins. We will also further characterize the nature of dots that contain RNAs retained near the site of transcription. Understanding these processes is critical for human health. It is estimated that at least 60% of the human genome undergoes alternative splicing, most of which probably occurs co-transcriptionally. This research therefore has relevance to public health, as splicing errors also generate aberrant proteins and are associated with important human genetic diseases, including cancer, muscular dystrophy, and Alzheimer's. In addition, the exosome - involved in RNA turnover and dot formation - is implicated in autoimmune disorders.